On average, 20% of corn produced in the U.S. is used for domestic food and industrial purposes including wet milling. In 1995, American farmers produced 7.4 billion bushels of corn, 20.6% of which was refined by the corn wet milling industry into more than 51 billion pounds of product.
Material output from the corn wet milling process includes starch for direct use or chemical modification, starch used as a degradative feedstock for the manufacture of an abundance of ancillary products, and coproducts/byproducts such as gluten feed, gluten meal and corn oil. As the list of products containing corn derived ingredients grows, so does the percentage of the U.S. crop that is utilized by the wet milling industry.
The central component critical in the direct, or indirect, use of corn for many products is starch. Interestingly, however, in some instances the unit value of coproducts exceeds that of starch. Such is the case with corn oil, having a value of approximately $0.23/lb, compared to $0.126/lb for starch (ERS 1995; ERS 1995). Corn wet millers rely on credits obtained from coproduct isolation and sale to minimize the net cost of the corn they grind for starch recovery. The monetary value realized by the isolation and sale of oil, or oil containing products (e.g., dry germ), is an important portion of these coproduct credits. Furthermore, because of the high unit value of oil, the coproduct credit for this material is more sensitive to changes in refining yield than all other coproducts.
In recognizing the importance and value of constituents isolated from grain in the wet milling process, it is useful to be aware of the starting composition of the grain and its parts. On a whole grain dry weight basis (db), corn is composed of the following primary constituents of economic importance: 4.0% oil, 9.7% protein, 69.8% starch, 3.5% sugars and 5.9% fiber. Similarly, the average composition of component parts of unprocessed grain is as follows: (1) the germ (defined as the organ inclusive of the scutellum and embryo proper) comprises 11.9% of the whole kernel and contains 34% oil, 8.2% starch, 18.8% protein, 10.8% sugar and 10.1% ash, and (2) the endosperm comprises 82% of the whole kernel and contains 86% starch, 9.4% protein, 0.8% oil and 0.6% sugar (Earle, F. R., J. J. Curtis, et al., "Composition of the Component Parts of the Corn Kernel"; Cereal Chem.; Vol. 23(5); pp. 504-511; 1946; incorporated herein in its entirety by reference). By calculation, Earle, et al., (1946), have determined that 84% of the seed oil is found in the germ and 98% of the kernel starch is located in the endosperm.
The purpose of the wet milling process is to fractionate the kernel and isolate chemical constituents of economic value into their component parts. This pertains specifically to starch, which is fractionated into a highly purified form. Other materials are typically isolated in crude forms (e.g., unrefined oil) or as a wide mix of materials which commonly receive little to no additional processing beyond drying. Hence, in the wet milling process grain is softened by steeping and cracked by grinding to release the germ from the kernels. The germ is separated from the heavier density mixture of starch, hulls and fiber by "floating" the germ segments free of the other substances in a centrifugation process. This allows a clean separation of the oil-bearing fraction of the grain from tissue fragments that contain the bulk of the starch. Since it is not economical to extract oil on a small scale, many wet milling plants ship their germ to large, centralized oil production facilities. Oil is expelled or extracted with solvents from dried germs and the remaining germ meal is commonly mixed into corn gluten feed (CGF), a coproduct of wet milling. Typical composition of spent germ cake, the germ material remaining after oil is extracted, is 20% starch, 25% protein, 1% fat, 10% crude fiber and 25% pentosans (Anderson, R. A. and S. A. Watson, "The Corn Milling Industry"; CRC Handbook of Processing and Utilization in Agriculture; A. Wolff, Boca Raton, Fla., CRC Press, Inc.; Vol. 11; Part 1; Plant Products: 31-61; 1982; incorporated herein in its entirety by reference). Hence, starch contained within the germ is not recovered as such in the wet milling process and is channeled to CGF. The unit value of CGF is roughly 20% that of corn oil and 50% that of corn starch. While increasing the oil content in seed, it is desirable that endosperm size and hence, starch content not be reduced because it is helpful if starch revenues are also maintained.
Current research indicates that genetic selection in maize can lead to increased oil content in the embryo, but that the resultant genotype is associated with reduced starch production. See e.g. Doehlert and Lanibert, "Metabolic Characteristics Associated with Starch, Protein, and Oil Deposition in Developing Maize Kennels"; Crop Sci.; Vol. 32; pp. 151-157; (1991); incorporated herein in its entirety by reference.
The central importance of starch to plant development and to food, feed, and industrial markets has motivated researchers across many years to look for mechanisms which control starch biosynthesis. Mutants of maize which affect seed starch deposition have been instrumental in characterizing the biochemistry of starch synthesis. Considerable research effort continues to explore the metabolic systems involved in synthesizing starch, but in addition molecular techniques are being used to dissect genes which encode enzymes known to be critical in starch biosynthesis. In discovering which regions of the genes encode metabolism-controlling aspects of the enzymes, scientists are beginning to manipulate starch metabolism through genetic engineering. Interest in controlling starch biosynthesis through molecular and genetic techniques has intensified significantly in recent years and several recent reviews describe fundamental aspects of starch biosynthesis and/or how they may be manipulated in transgenic plants, see e.g. Hannah, L. C., M. Giroux et al, "Biotechnological Modification of Carbohydrates for Sweet Corn and Maize Improvement"; Scientia Horticulturae; Vol. 55; pp. 177-197; 1993; Smith, A. M. and C. Martin, "Starch Biosynthesis and the Potential for its Manipulation"; Biosyn. And Manipulation of Plant Products; D. Grierson; Vol. 3; pp. 1-54; 1993; Visser, R. G. F. and E. Jacob, "Towards Modifying Plants for Altered Starch Content and Composition"; Trends In Biotechnology; Vol. 11; pp. 63-68; 1993; Muller-Rober, B. and J. Kossmann, "Approaches to Influence Starch Quantity and Starch Quality in Transgenic Plants"; Plant Cell Environ.; Vol. 17; pp. 601-613; 1994; Bhullar, S. S., "Bioregulation of Starch Accumulation in Developing Seeds"; Current Science; Vol. 68(5); pp. 507-516; 1995; Morell, M. K., S. Rahan, et al., "The Biochemistry and Molecular Biology of Starch Synthesis in Cereals"; Aust. J. Plant Physiol.; Vol. 647-660; 1995; Nelson, O. and D. Pan, "Starch Synthesis in Maize Endosperms"; Plant Physiol.; Vol. 46; pp. 475-496; 1995; Wasserman, et al., "Biotechnology: Progress Toward Genetically Modified Starches" Cereal Foods World; Vol. 40(11); pp. 810-817; 1995; all incorporated herein in its entirety by reference.
Sucrose is considered to be the primary metabolite utilized in the synthesis of starch, although seed grown in vitro with the reducing sugars, glucose or fructose, also produce starch. In simple terms, the sugars are converted into the sugar nucleotides, ADP-glucose and UDP-glucose, either directly or via phosphorylated carbohydrate intermediates. The sugar nucleotides are substrates for the synthase enzymes which polymerize the glucosyl portion of the molecules into long chains of glucose. The polymers remain essentially linear (amylose) or become branched (amylopectin) and combine in a specific fashion to become granules of starch. The mechanism of initiating the synthesis of the glucosyl polymer is not known with certainty, although it is hypothesized that a protein, amylogenin, may serve a nucleating role in the process (Singh, D. G., J. Lomako, et al., ".beta.-glucosylarginine: a New Glucose-Protein Bond in a Self-glucosylating Protein from Sweet Corn"; FEBS Lett.; Vol. 376; pp. 61-64; 1995; incorporated herein in its entirety by reference).
The use of seed starch mutants in various crop plants (e.g., corn, pea) and the production of transgenic plants which over- or underexpress specific proteins has indicated that many proteins/enzymes are capable of affecting starch biosynthesis in storage organs. This can occur directly, by impacting the proteins which: (1) produce the substrate(s) for starch synthesis (2) initiate the glucose polymerization process and elongate the structure into macromolecules, or (3) alter the structure of the polymers once the elongation process has begun. Enzymes known, or believed, to participate in these processes include, but are not limited to, ADP-glucose and UDP-glucose pyrophosphorylases, bound and soluble starch eases, starch phosphorylases, starch granule bound and soluble branching enzymes, debranching enzymes, isoamylases and disproportionating enzymes. In addition to a direct impact on starch metabolism, starch production can also be negatively impacted by dysfunction or deficiency of proteins which are catalysts in sugar metabolism or act as transporters of intermediary compounds. Examples of those involved in sugar metabolism include, but are not limited to, sucrose synthases, sucrose phosphate synthase, sucrose phosphate phosphorylase, hexokinases, phosphoglucomutases and phosphoglucoisomerases. Proteins involved in assimilate transport, such as the brittle-1 protein of maize endosperm amyloplast membranes (Cao, H. P., T. D. Sullivan, et al., "Bt1, a Structural Gene for the Major 39-44 kDa Amyloplast Membrane Polypeptides"; Physiol. Plant; Vol. 95(2); pp. 177-186; 1995; Shannon, J. C., F. M. Pien, et al., "Nucleotides and Nucleotide Sugars in Developing Maize Endosperms-Synthesis of ADP-glucose in Brittle-1"; Plant Physiology; Vol. 110(3); pp. 835-843; 1996; both incorporated herein in its entirety by reference), sucrose carrier proteins (Weig, et al., "An Active Sucrose Carrier (Scr1) that is Predominantly Expressed in the Seedlings of Ricinus Communis L."; Journal of Plant Physiology; Vol. 147(6); pp. 685-690; (1990); incorporated herein in its entirety by reference) or others homologous to the hexose transporter of the chloroplast (Fitzpatrick, et al., "The Hexose Translocator of the Chloroplast Envelope"; J. Exp. Bot.; Vol. 47; pp. 79; (1996); incorporated herein in its entirety) can also affect starch synthesis by restricting the availability of substrates for normal starch and/or sugar metabolism.
One enzyme which has been shown to be particularly important in starch biosynthesis, as well as in the synthesis of bacterial glycogen, is ADP-glucose pyrophosphorylase (AGP). In bacteria, AGP is a homotetrameric protein, while in plants it is a heterotetrameric complex of two different protein subunits. Dysfunction or absence of either subunit severely reduces starch synthesis. The starch mutants of maize which affect the AGP subunits in the endosperm are brittle-2 (bt2; small subunit) and shrunken-2 (sh2; large subunit) (Giroux, M. and L. Hannah; "ADP-glucose Phyrophosphorylase in Shrunken-2 and Brittle-2 Mutants of Maize"; Mol. Gen. Genet.; Vol. 243; pp. 400-408; 1994; incorporated herein in its entirety by reference). The reduction of accumulated starch coincident with lower AGP activity in mutant pea embryo (Hylton. C. and A. M. Smith, "The rb Mutation of Peas Causes Structural and Regulatory Changes in ADP-glucose Pyrophosphorylase from Developing Embryos"; Plant Physiol.; Vol. 99; pp. 1626-1634; 1992; incorporated herein in its entirety by reference), Arabidopsis leaf (Neuhaus, H. E. and M. Stitt, "Control Analysis of Photosynthate Partitioning. Impact of Reduced Activity of ADP-glucose Pyrophosphorylase or Plastid Phosphoglucomutase on the Fluxes to Starch and Sucrose in Arabidopsis thaliana (L.) Heynh" Planta; Vol. 182; pp. 445-454; 1990; incorporated herein in its entirety by reference), or in the tuber of antisensed potato plants (Muller-Rober, B., U. Sonnewald, et al., "Inhibition of the ADP-Glucose Pyrophosphorylase in Transgenic Potatoes Leads to Sugar-Storing Tubers and Influences Tuber Formation and Expression of Tuber Storage Protein Genes"; EMBO; Vol. 11(4); pp. 1229-1238; 1992; incorporated herein in its entirety by reference) demonstrates the commanding role of AGP in controlling starch deposition in a wide variety of tissues and plants.
Numerous genes encoding the small and large subunits of AGP from plants have been described (Smith-White, B. J. and J. Preiss, "Comparison of Proteins of ADP-Glucose Pyrophorylase from Diverse Sources"; J. Mol. Evol.; Vol. 34; pp. 449-464; 1992; incorporated herein in its entirety by reference). The corresponding genes which have been described for maize are the endosperm specific Bt2 and Sh2 genes (Bae, J. M., M. Giroux, et al., "Cloning and Characterization of the Brittle-2 Gene of Maize"; Maydica; Vol. 35; pp. 317-322; 1990; Bhave, M. R, S. Lawrence, et al., "Identification and Molecular Characterization of Shrunken-2 cDNA Clones of Maize"; Plant Cell; Vol. 2; pp. 581-588; 1990; incorporated herein in its entirety by reference) and AGP1 (Giroux, M. and B. Smith-White, et al., "The Large Subunit of the Embryo Isoform of ADP Glucose Pyrophosphorylase from Maize"; Plant Physiol.; Vol. 108; pp. 1333-1334; 1995; incorporated herein in its entirety by reference). Although referred to as an embryo isoform because of its predominance in the germ of the seed, AGP1 is also expressed in the endosperm (Giroux, M. and L. Hannah, "ADP-Glucose Pyrophosphorylase in Shrunken-2 and Brittle-2 Mutants of Maize"; Mol. Gen. Genet.; Vol. 243; pp. 400-408; 1994; incorporated herein in its entirety by reference). AGP1 represents the large subunit of the embryo isoform, whereas AGP2, to-date an uncharacterized gene, corresponds to the small subunit (Giroux and Hannah, 1994).
AGP is an allosteric enzyme and in plants is activated by 3-phosphoglyceric acid (3-PGA) and inhibited by inorganic phosphate (Pi) to varying degrees, depending upon the species and organ source (Preiss, J. "Biosynthesis of Starch: ADP-Glucose Pyrophosphorylase, the Regulatory Enzyme of Starch Synthese: Structure-Function Relationships"; Denpun Kagaku; Vol. 40(2); pp. 117-131; 1993; incorporated herein in its entirety by reference). The importance of the allosteric properties of AGP is recently demonstrated when transgenic plants expressing a bacterial form of AGP (glgC16), which is allosterically "deregulated" compared to the native plant AGP, accumulated 35% more starch than controls (Stark, D. M., K. P. Timmerman, et al., "Regulation of the Amount of Starch in Plant Tissues by ADP-Glucose Pyrophosphorylase"; Science; Vol. 258; pp. 287-292; 1992; incorporated herein in its entirety by reference). Furthermore, allosteric modification of the maize native Sh2 gene can increase seed weight, presumably due, at least in part, to an affect on starch deposition (Giroux, M. J., J. Shaw, et al., "A Single Gene Mutation that Increases Maize Seed Weight"; Proc. Natl. Acad. Sci.; Vol. 93; pp. 5824-5829; 1996; incorporated herein in its entirety by reference). Allosteric variants of AGP, which are less responsive to 3-PGA and Pi are reported to occur naturally in plants (Kleczkowski, L. A., P. Villand, et al., "Insensitivity of Barley Endosperm ADP-Glucose Pyrophosphorylase to 3-Phosphoglycerate and Orthophosphate Regulation"; Plant Physiol.; Vol. 101; pp. 179-186; 1993; incorporated herein in its entirety by reference) and they also can be engineered into plants through gene manipulation and plant transformation.
Because maize germ is known for its oil storing capacity, starch synthesis in this organ has been studied much less than comparable metabolism in the endosperm. Starch granules of the germ are morphologically distinct from those of the endosperm (Adkins, G. K. and C. T. Greenwood, "The Isolation of Cereal Starches in the Laboratory"; Starch; Vol. 7; pp. 213-218; 1966; incorporated herein in its entirety by reference), but in all probability the metabolic differences in endosperm and germ starch biosynthesis are not fundamentally extensive. In fact, many of the enzyme activities important in endosperm sugar and starch metabolism are also very active in the germ (Lee, E. Y. C.; "Multiple Forms of 1,4-a-Glucan Phosphorylase in Sweet Corn"; FEBS Letters; Vol. 27(2); pp. 341-345; 1972; Doehlert, D., T. Kuo, et al., "Enzymes of Sucrose and Hexose Metabolism in Developing Kernels of Two Inbreds of Maize"; Plant Physiol.; Vol. 86; pp. 1013-1019; 1988; both incorporated herein in its entirety by reference). More specifically, AGP activity is known to occur in maize germ (Tsai, C. and O. Nelson, "Starch-Deficient Maize Mutant Lacking Adenosine Diphosphate Glucose Pyrophosphorylase Activity"; Science; Vol. 151; pp. 341-343; 1966; Dickinson, D. B. and J. Preiss, "Presence of ADP-glucose Pyrophosphorylase in Shrunken-2 and Brittle-2 Mutants of Maize Endosperm"; Plant Physiol.; Vol. 44; pp. 1058-1062; 1969; both incorporated herein in their entirety by reference), although it follows a different developmental profile than AGP activity in the endosperm (Prioul, J. L., E. Jeannette, et al., "Expression of ADP-glucose Pyrophosphorylase in Maize (Zea mays L.) Grain and Source Leaf During Grain Filling"; Plant Physiol.; Vol. 104; pp. 179-187; 1994; incorporated herein in its entirety by reference). An analogous situation is expected to occur for starch.
Based on the foregoing, there is a need to provide seed with an increased concentration of coproducts such as oil.
It is therefore an object of the present invention to provide seeds with an increased concentration of oil.
It is a further object of the present invention to provide seeds with an increased concentration of oil compared to the wild type without an increase in seed weight or endosperm size.
It is a further object of the present invention to provide seeds with an increased concentration of oil compared to the wild type.
It is a further object of the present invention to provide methods of producing seeds with an increased concentration of oil without a reduction in endosperm size.
It is a further object of the present invention to provide methods of producing seeds with an increased concentration of oil without an increase in seed weight.
It is a further object of the present invention to provide methods of producing seeds with an increased concentration of oil without the resulting genotype being associated with reduced starch production in the endosperm.